Hematopoietic cell transplantation (HCT) can cure genetic blood disorders through the replacement of defective blood cells by healthy cells. However, life-threatening complications limit the use of HCT therapy in the clinic. Complications in the recipient result from toxic conditioning and from graft-versus-host disease (GVHD), a condition where the donor T-cells attack the recipient (host) tissues. Complete, prolonged depletion of both donor and host T cells permits stable engraftment of donor cells using reduced-intensity conditioning in rodents, without GVHD. However, complete T cell depletion is difficult to achieve in humans and infections, hematopoietic failure, and GVHD remain major complications of clinical HCT. Similar to outcomes in humans, miniature swine develop severe GVHD, infectious complications, or hematopioetic failure following standard clinical myeloablative conditioning and HCT. The investigators recently developed a novel, mild, preclinical protocol for HCT which permits donor cells to engraft without GVHD. Indeed, the treatment of miniature swine with a low-dose of total body irradiation, partial T-cell depletion, and a short course of cyclosporine A has reliably established donor HC engraftment without GVHD despite infusion of a very high dose of T-cell replete haploidentical (e.g., parent-into-child) HCT. Surprisingly, additional treatment with donor leukocyte infusion (DLI), a procedure known to ensure full replacement of HC in engrafted rodents, did not result in full (donor) hematopoietic replacement. The investigators surmise that the persistence of donor and recipient T cells in treated pigs promotes HC engraftment, inhibits GVHD, but also hinders the DLI conversion effect. Indeed, preliminary data suggest that regulatory cells persist in treated animals and inhibit both GVHD and DLI alloreactivity. Thus, the goal of this project is to elucidate the immunological mechanisms responsible for facilitating engraftment without GVHD, and to test new strategies to restore the DLI conversion effect in swine. The investigators hypothesize that immune modulation with regulatory cell involvement promotes engraftment and inhibits GVHD and DLI effects by inactivating donor, recipient, and DLI alloreactive T-cells. This hypothesis will be tested by the following specific aims, to: 1) elucidate the immunological mechanisms controlling GVH and HVG alloreactive T cells in vivo following HCT; and 2) restore the ability of DLI to enhance donor engraftment levels without GVHD through specific immune modulation. This project is innovative in that it investigates approaches for HCT that harness the immunomodulatory capacities of the immune system, rather than immune ablation, to promote better engraftment with fewer complications. The long-term goal of these studies is to translate promising protocols for parent-into-child HCT and DLI to the clinic. Successful translation would provide a cure for many screenable genetic disorders including hemoglobinopathies, lysosomal storage diseases, inherited aplastic anemias, and primary immunodeficiency diseases. If the aims of this application are achieved, these results could have a major impact on clinical HCT approaches. RELEVANCE: Toxicities and complications associated with hematopoietic cell transplantation (HCT) currently limit this curative therapy for genetic blood disorders in the clinic. The goal of this application is to understand the immunological mechanisms responsible for facilitating high-level haploidentical (e.g., parent into child) donor hematopoietic cell engraftment without complications. The long-term goal of these studies is to translate promising nontoxic HCT protocols using parental donors to replace diseased blood cells and cure genetic blood disorders in children.